The goal of this proposal is to elucidate the role of specific host proteins that interact with HIV-1 Gag in virus replication. We have identified the host proteins kindlin-3 and pacsin2 as binding partners for the NC and p6 domains of HIV-1 Gag, respectively. Knockdown and reconstitution experiments show that both proteins are critical for the spreading of HIV-1 among T cells. Based on our preliminary data, our hypothesis is that kindlin-3 and pacsin2 are involved in the cell-to- cell transmission of HIV-1. Kindlin-3 is required for the function of integrins such as LFA-1, which has been implicated in HIV transmission. However, our data demonstrate a striking requirement for kindlin-3 for HIV-1 replication even in T lymphoid cells lacking LFA-1. Our working model is that HIV-1 Gag mimics the kindlin-recruiting activity of integrins to induce donor cell polarization towards the virologial synapse and thereby promote virus transfer. We propose to investigate the mechanism by which kindlin-3 associates with HIV-1 virions, which step of the viral life cycle is promoted by kindlin-3, whether downstream partners of kindlin-3 are involved in HIV-1 replication, and whether the kindlin-3-dependent activity of integrins other than LFA-1 is important for HIV-1 replication. Pacsin2 can generate membrane curvature and nucleate filopodia formation. HIV-1 frequently buds from filopodia, and viral filopodia capped by Gag have been implicated in HIV-1 cell-cell transmission. Thus, pacsin2 may be important for HIV-1 transmission because it is involved in viral filopodia formation. We propose to investigate the role of Gag ubiquitination in the recruitment of pacsin2 into viral particles, the precise role of pacsin2 in HIV-1 replication, he role of the actin remodeling activity of pacsin2 in HIV-1 replication, and whether pacsin2 interaction partners on recycling endosomes are involved in HIV-1 replication. The transfer of HIV-1 from cell to cell is thought to be the predominant mode of transmission, but the molecular basis is poorly understood. An understanding of the roles of kindlin-3 and pacsin2 in HIV-1 replication may provide novel insights into this important mode of transmission.